HIV Nef ubiquitination and its function (Project Summary) We have made an important discovery that both HIV Nef and SIV Nef are multiple ubiquitinated and the Nef ubiquitination, specifically the di-ubiquitination of the lysine 144 in HIV-1 Nef is required for Nef downregulation of CD4, the primary receptor of HIV infection. Ubiquitination is the attachment of a 76 aa ubiquitin molecule to a substrate protein. It is well established that the ubiquitination is the sorting signals for protein degradation in proteasome, for receptor endocytosis and for protein intracellular trafficking. Since the major Nef pathological activity is the downregulation of cell surface CD4 and rerouting MHC-1 intracellular trafficking from TGN to the endosomes, the Nef ubiquitination could be the signals required for Nef/target complexes to enter endosomal/lysosomal degradation pathway. Our finding thus suggests that HIV-1 adopts a strategy to overcome the host defense by diverting host defense proteins into the ubiquitination/endosomal/lysosomal degradation pathway. This strategy differs from the known viral strategy of destroying host defense proteins through the ubiquitination/proteasome pathway. Therefore our discovery may open up a whole new field of HIV biology research. The proposed studies (R21) address the following questions: Besides CD4 downregulation, do other Nef functions also require Nef ubiquitination? After Nef ubiquitination, what are the steps that lead to the endosomal targeting and the lysosomal degradation? How does Nef ubiquitination affect the HIV-1 infectivity? In Aim 1, we will further characterize the ubiquitin attachment sites in both HIV-1 and SIV Nef since the ubiquitination at different lysine residues may be responsible for different sorting signals. To identify the Ub-Nef binding proteins (Ub-receptors), we will use pull down assay, HPLC analysis and yeast-two hybrid assay. In Aim 2, we will examine the ubiquitination-deficient Nef mutants that we have identified in inducing the downregulation and lysosomal degradation of CD4 and MHC-I. To evaluate the role of Nef ubiquitination in HIV-1 infectivity, we will generate pNL4-3 provirion DNA encoding the HIV-1 defective in Nef ubiquitination. The infectivity of the pNL4-3 mutants will be evaluated by the single-round infectivity assay and viral replication assay. PUBLIC HEALTH RELEVANCE: Nef plays a crucial role in the pathogenicity of HIV-1 and the development of AIDS. Our discovery of Nef ubiquitination will open up a new field of HIV biology research and provide a new specific target for anti-HIV therapy.